Fluorescence measurements are employed in a variety of analyses and in such techniques, illumination of a first wavelength absorbed by a sample induces the sample to emit light of a second wavelength. The wavelength and/or intensity of the secondary emission may be correlated with composition, concentration, physical environment and similar parameters. In one particular class of fluorescence analyses, cells of various tissue types are grown in culture and incubated in a growth medium with a fluorescent dye. The cells will absorb the dye at particular rates, and these rates may be correlated with various physiological functions of the cells such as K.sup.+ channel activity. A cell which has absorbed dye will typically fluoresce at an enhanced intensity as compared to the growth medium which incorporates that dye. Fluorescent analyses of this type are of significant importance in the pharmaceutical industry since they may be employed to screen a variety of tissue types for interaction with chemical species of pharmaceutical interest.
In an analysis of this type, cells are cultured in a multiple well plate. These plates typically include 48 or 96 wells, each of which comprises a cylinder of approximately 5 millimeters in diameter, closed at one end by an optically transparent bottom surface and open at the other. The cells are cultured in a layer on the bottom surface of the wells with a supernatant layer of growth medium thereabove. Chemical species being assayed are placed into the supernatant liquid together with a fluorescent dye and the effect of the chemical species on cell metabolism is assayed by measuring the fluorescence of the cell layers. Such techniques are well known in the art and are described, for example, in U.S. Pat. Nos. 4,343,782, 4,835,103 and PCT published application WO 90/15317.
In order to measure the fluorescence of the cells, the cell layers are illuminated with light of a first wavelength and emission at a second wavelength is monitored by a photodetector device. Problems arise in this type of an assay because the cell layer is typically on the order of 10 microns in thickness, while the depth of the supernatant liquid is on the order of many millimeters. While the relative intensity of the emission from the supernatant liquid is generally lower than that from the cells which have absorbed the dye, fluorescence from the supernatant liquid constitutes a significant source of error in these assays because of the large volume of the supernatant.
This problem is illustrated by the drawing of FIG. 1 which depicts a particular prior art methodology for fluorescence measurements. Shown in the figure is a well 10 which is typically a part of a larger plate comprising an array of such wells. Disposed within the well 10 is a volume of cell growth medium 12, and a layer of cultured cells 14 is shown atop the bottom surface 16 of the well 10. In the illustrated embodiment, the layer of cells 14 is flooded with illumination 18 which induces fluorescence in the cells 14 as well as in the supernatant liquid 12. A detector 20, typically operating in connection with a lens 22 views the cell layer 14. Generally, the detector 20 and source of the light beam 18 (not shown) are on a common optical axis and share some optical elements such as the lens 22. It will be appreciated from the figure that fluorescence in the supernatant liquid 12 will significantly interfere with the measurement of cell fluorescence.
In order to overcome problems of background fluorescence, the art has employed a microscope to measure the fluorescence of cell layers disposed in a well with a supernatant liquid. The limited depth of focus of the microscope minimizes background fluorescence contributions. FIG. 2 depicts one such prior art approach. As shown therein, a well 10 includes a supernatant growth medium 12 and a cell layer 14 upon the bottom 16 thereof. A microscope, indicated schematically by lens 24 is positioned to view the cell layer 14. It operates in conjunction with a detector 20 to measure the fluorescence of a very small area 26 of the cell layer 14; and as mentioned previously, the source of illumination is generally incorporated into the microscope/detector unit. The extremely limited depth of focus of the microscope 24 permits the detector to "see" only a limited portion of the supernatant liquid and hence only a small portion of the fluorescence therein, indicated by region 28 in the figure, contributes to background noise.
While the approach of FIG. 2 provides acceptable accuracy, the severely restricted field of view of the microscope greatly limits the speed of this technique. A microscope of 40.times. will give a depth of field of 50 microns, which is adequate to minimize background; but, the field of view of the microscope will be approximately 10 microns. In order to obtain an accurate signal characteristic of K.sup.+ channel activity it is necessary to scan a several hundred square micron portion of each well, typically in a raster pattern, to develop a statistically significant signal. Because of the limited field of view of the microscope, the scan takes several minutes. In many studies, it is desirable to update data every few minutes; therefore, the system is limited to measuring a single well over the course of an experiment which may run several hours. Even if only a single scan is made per well, read times for a 96 well plate will be several hours. In addition to the foregoing, microscope arrangements of this type are quite expensive. U.S. Pat. No. 5,097,135 discloses the use of a microscope for making fluorescence measurements of this type. U.S. Pat. No. 5,091,652 discloses a microscope based fluorescence analyzer which operates in a scanning mode.
Thus, it will be seen that there is a need for a fluorescence analyzer which is capable of measuring the fluorescence of a thin cell layer disposed in a well of a multiple well plate, with minimal interference from fluorescence in a supernatant medium. It is further desirable that the analyzer be capable of rapidly measuring the fluorescence of a plurality of samples. It is most desirable that the analyzer be capable of making a large number of measurements in parallel. The present invention, as will be apparent, from the drawings, discussion and description which follow, provides for an improved apparatus and methodology for fluorescence measurements. The present invention minimizes background fluorescence and may be operated to measure, in parallel, the fluorescence of cell layers in each of the wells of a multiple well plate. These and other advantages of the present invention will be readily apparent from the drawings, discussion and description which follow.